Berkeley Lab Scientists Reveal Path to Protein Crystallization

By assembling a crystalline envelope around a cell, surface-layer (S-layer)
proteins serve as the first point of contact between bacteria, extremophiles
and other types of microbes and their environment.

Now, scientists at the Molecular Foundry, a nanoscience user facility at Berkeley
Lab, have used atomic force microscopy to image in real time how S-layer
proteins form crystals in a cell-like environment. This direct observation of
protein assembly could provide researchers with insight into how microorganisms
stave off antibiotics or lock carbon dioxide into minerals.

“Many proteins self-assemble into highly ordered structures that provide
organisms with critical functions, such as cell adhesion to surfaces, transformation
of CO2 into minerals, propagation of disease, and drug resistance,” said
James DeYoreo, Deputy Director of the Molecular Foundry. “This work is
the first to provide a direct molecular-level view of the assembly pathway in
vitro. Once this knowledge can be extended to assembly in a living system, it
may lead to strategies for capitalizing on or interfering with these functions.”

Unraveling the pathway for S-layer formation allows scientists to investigate
how bacteria or other microbes negotiate interactions with their environment.
DeYoreo and colleagues employed in situ atomic force microscopy—a probe
technique used to study a crystal’s surface in its natural setting with
atomic precision—to watch S-layer proteins assemble from solution onto
a flat, biological membrane called a lipid bilayer. Unlike classical crystal
growth, in which atoms form into ordered ‘seeds’ and grow in size,
the team showed S-layer proteins form unstructured blobs on the bilayers before
transforming into a crystalline structure over the course of minutes.

“We can actually see these proteins from solution sticking and arranging
on the lipid bilayers where they spontaneously condense into many protein blobs—then,
minutes later, they transform into a crystalline structure with a square lattice
of tetramers,” said Sungwook Chung, a staff scientist in the Physical
Biosciences Division and user at the Molecular Foundry. “This is an important
discovery as it gives direct evidence for a multi-stage assembly pathway with
an intermediate, amorphous phase forming before folding into a two-dimensional,
crystalline array.”

Foundry post-doctoral researcher Seong-Ho Shin, working with Foundry Director
Carolyn Bertozzi, says understanding how S-layers interact with their environment
could help in recognizing how organisms resist antibacterial drugs, or how microbes
turn carbon dioxide into solid carbonates. Among the first protein structures
to be used to organize nanostructures, S-layers are also attractive scaffold
template materials for growing or organizing nanowires or quantum dots.

In a study inspired by this work, Foundry staff scientist Steve Whitelam used
computer modeling to investigate crystallization pathways of model S-layer proteins.
As model proteins encounter one another, they might stick together at any angle
at which they collide (mediated by non-specific interactions), or bind in the
correct orientation needed to form a crystal (mediated by directional interactions).

By adjusting these interactions, Whitelam identified parameter regimes within
which unstructured blobs of proteins form prior to crystallization. Searching
through parameter space, he found crystals often form more reliably if model
proteins interacted directionally and nonspecifically, rather than through directional
interaction alone. What’s more, he adds, these findings may apply to different
kinds of materials.

“Many biological and inorganic materials assemble and crystallize through
intermediate phases that are often amorphous,” said Whitelam, who is in
the Foundry’s Theory of Nanostructured Materials Facility. “Developing
a molecular model of crystallization in a particular system helps us to understand
mechanisms of crystallization in general.”

Two papers reporting this research titled, “Self-catalyzed growth of
S-layers via an amorphous-to-crystalline transition limited by folding kinetics,”
(Chung, Shin, Bertozzi, DeYoreo) appearing in Proceedings of the National Academy
of Sciences and “Control of pathways and yields of protein crystallization
through the interplay of nonspecific and specific attractions,” (Whitelam)
appearing in Physical Review Letters, are available online at (http://www.pnas.org/content/early/2010/09/01/1008280107)
and (http://prl.aps.org/abstract/PRL/v105/i8/e088102)

These works were performed at the Molecular Foundry and supported by DOE’s
Office of Science.

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